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• W2093643472 abstract "Leukemia inhibitory factor (LIF) is a polyfunctional cytokine known to require at least two distinct receptor components (LIF receptor α-chain and gp130) in order to form a high affinity, functional receptor complex. In this report, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100,000, which is similar to a previously described form of soluble gp130 in human serum, and a previously undescribed small form with a molecular weight of approximately 50,000. Using a panel of monoclonal antibodies raised against the extracellular domain of human gp130, we were able to show that the small form of the urinary gp130 probably contained only the hemopoietin domain. Both forms of gp130 bound LIF specifically and were capable of forming heterotrimeric complexes with soluble human LIF receptor α-chain in the presence of human LIF. In addition to the soluble forms of gp130, a soluble form of LIF receptor α-chain was also detected in human urine and plasma. Leukemia inhibitory factor (LIF) is a polyfunctional cytokine known to require at least two distinct receptor components (LIF receptor α-chain and gp130) in order to form a high affinity, functional receptor complex. In this report, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100,000, which is similar to a previously described form of soluble gp130 in human serum, and a previously undescribed small form with a molecular weight of approximately 50,000. Using a panel of monoclonal antibodies raised against the extracellular domain of human gp130, we were able to show that the small form of the urinary gp130 probably contained only the hemopoietin domain. Both forms of gp130 bound LIF specifically and were capable of forming heterotrimeric complexes with soluble human LIF receptor α-chain in the presence of human LIF. In addition to the soluble forms of gp130, a soluble form of LIF receptor α-chain was also detected in human urine and plasma. Leukemia inhibitory factor (LIF) 1The abbreviations used are: LIF, leukemia inhibitory factor; hLIF, human LIF; LIFRα, LIF receptor α-chain; hLIFRα, human LIFRα; shLIFRα, soluble human LIFRα; shgp130, soluble human gp130; sshgp130, short form of soluble human gp130; FN III, fibronectin type III; STAT-3, signal transducer and activator of transcription 3; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CNTF, ciliary neurotrophic factor; OSM, oncostatin-M; IL, interleukin; IL-6Rα, interleukin 6 receptor α-chain; BS3, bis(sulfosuccinimidyl)suberate. 1The abbreviations used are: LIF, leukemia inhibitory factor; hLIF, human LIF; LIFRα, LIF receptor α-chain; hLIFRα, human LIFRα; shLIFRα, soluble human LIFRα; shgp130, soluble human gp130; sshgp130, short form of soluble human gp130; FN III, fibronectin type III; STAT-3, signal transducer and activator of transcription 3; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CNTF, ciliary neurotrophic factor; OSM, oncostatin-M; IL, interleukin; IL-6Rα, interleukin 6 receptor α-chain; BS3, bis(sulfosuccinimidyl)suberate. is a polyfunctional cytokine that can act on a wide range of cell types including osteoblasts, hepatocytes, adipocytes, neurons, embryonal stem cells, and megakaryocytes (1Nicola N.A. Hilton D.J. LeRoith D. Bondy C. Growth Factors and Cytokines in Health and Disease. 2B. JAI Press, Greenwich, CT1996: 605-660Google Scholar). LIF exerts its multiple biological functions through a specific cell surface receptor system, which consists of at least two membrane-bound glycoproteins, the LIF-binding chain (LIFRα) and gp130. LIF binds first to LIFRα with low affinity (2Gearing D. Thut C.J. VandenBos T. Gimpel S.D Delaney P.B. King J. Price V. Cosman D. Beckmann M.P. EMBO J. 1991; 10: 2839-2848Crossref PubMed Scopus (516) Google Scholar) and then to gp130 to form a high affinity functional receptor complex leading to activation of downstream signal transduction pathways (3Gearing D.P. Comeau M.R. Friend D.J. Gimpel S.D. Thut C.J. McGourty J. Brasher K.K. King J.A. Gillis S. Mosley B. Ziegler S.F. Cosman D. Science. 1992; 255: 1434-1437Crossref PubMed Scopus (793) Google Scholar, 4Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (610) Google Scholar, 5Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (591) Google Scholar, 6Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1246) Google Scholar). Both LIFRα and gp130 are members of the hemopoietin or cytokine type I family of receptors (7Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1877) Google Scholar, 8Cosman D. Cytokine. 1993; 5: 95-106Crossref PubMed Scopus (267) Google Scholar). The extracellular domains of members of this receptor family share common structural features including hemopoietin domains characterized by four conserved cysteine residues and a WSXWS motif and three fibronectin type III (FN III) modules (7Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1877) Google Scholar, 8Cosman D. Cytokine. 1993; 5: 95-106Crossref PubMed Scopus (267) Google Scholar). The membrane-bound gp130 was initially defined as the signal transducer of the interleukin-6 (IL-6) receptor system (9Taga T. Hibi M. Hirata Y. Yamasaki K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1189) Google Scholar, 10Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1092) Google Scholar) and has been shown subsequently to also be a component of the functional receptor complexes of ciliary neurotrophic factor (CNTF) (4Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (610) Google Scholar), oncostatin-M (OSM) (3Gearing D.P. Comeau M.R. Friend D.J. Gimpel S.D. Thut C.J. McGourty J. Brasher K.K. King J.A. Gillis S. Mosley B. Ziegler S.F. Cosman D. Science. 1992; 255: 1434-1437Crossref PubMed Scopus (793) Google Scholar, 11Gearing D.P. Bruce A.G. New Biol. 1992; 4: 61-65PubMed Google Scholar), cardiotrophin-1 (12Pennica D. King K. Shaw K.J. Luis E. Rullamas J. Luoh S.M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (498) Google Scholar, 13Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), and interleukin (IL)-11 (IL-11) (14Yin T. Taga T. Tsang M.L. Yasukawa K. Kishimoto T. Yang Y.C. J. Immunol. 1993; 151: 2555-2561PubMed Google Scholar, 15Fourcin M. Chevalier S. Lebrun J.J. Kelly P. Pouplard A. Wijdenes J. Gascan H. Eur. J. Immunol. 1994; 24: 277-280Crossref PubMed Scopus (50) Google Scholar, 16Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 4765-4775Crossref PubMed Scopus (253) Google Scholar). In addition to the cell membrane-anchored forms of LIFRα and gp130, it has been reported that naturally occurring soluble forms of these receptor molecules are present in biological fluids and may act as natural inhibitors of LIF activity (17Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar, 18Yamaguchi-Yamamoto Y. Tomida M. Hozumi M. Leukemia Res. 1993; 17: 515-522Crossref PubMed Scopus (15) Google Scholar, 19Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). We and others have shown previously that a soluble form of the mouse LIFRα with a molecular weight (M r) of approximately 90,000–150,000 occurs at high levels in normal mouse serum and is elevated dramatically during pregnancy (17Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar, 18Yamaguchi-Yamamoto Y. Tomida M. Hozumi M. Leukemia Res. 1993; 17: 515-522Crossref PubMed Scopus (15) Google Scholar). Recently, we have provided evidence that the soluble form of mouse LIFRα probably arises from an alternative splicing event of the LIFRα mRNA (20Owczarek C.M. Layton M.J. Robb L.G. Nicola N.A. Begley C.G. J. Biol. Chem. 1996; 271: 5495-5504Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Despite the high levels of soluble LIFRα in mouse serum, its analogue was not detected in human serum (17Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar). In contrast, a soluble form of gp130 with a M rof 90,000–110,000 has been found in human serum (19Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). Although gp130 functions as the high affinity converting and signaling subunit in the receptor complexes for IL-6, LIF, OSM, CNTF, cardiotrophin-1, and IL-11, OSM was the only cytokine in this family initially demonstrated to bind to membrane-bound gp130 (3Gearing D.P. Comeau M.R. Friend D.J. Gimpel S.D. Thut C.J. McGourty J. Brasher K.K. King J.A. Gillis S. Mosley B. Ziegler S.F. Cosman D. Science. 1992; 255: 1434-1437Crossref PubMed Scopus (793) Google Scholar), and subsequently, it has been shown that OSM can bind directly to the soluble form of gp130 with low affinity (21Sporeno E. Paonessa G. Salvati A.L. Graziani R. Delmastro P. Ciliberto G. Toniatti C. J. Biol. Chem. 1994; 269: 10991-10995Abstract Full Text PDF PubMed Google Scholar, 22Modrell B. Liu J. Miller H. Shoyab M. Growth Factors. 1994; 11: 81-91Crossref PubMed Scopus (19) Google Scholar). We and others have recently shown that the soluble form of gp130 was able to bind not only directly and specifically to OSM but also to LIF (22Modrell B. Liu J. Miller H. Shoyab M. Growth Factors. 1994; 11: 81-91Crossref PubMed Scopus (19) Google Scholar, 23Zhang J.G. Owczarek C.M. Ward L.D. Howlett G.J. Fabri L.J. Roberts B.A. Nicola N.A. Biochem. J. 1997; 325: 693-700Crossref PubMed Scopus (33) Google Scholar). Using biosensor technology, we were able to determine that the interaction between hLIF and soluble human gp130 was of low affinity, with an equilibrium dissociation constant of approximately 44 nm (23Zhang J.G. Owczarek C.M. Ward L.D. Howlett G.J. Fabri L.J. Roberts B.A. Nicola N.A. Biochem. J. 1997; 325: 693-700Crossref PubMed Scopus (33) Google Scholar). This low affinity interaction could explain previous failures in detecting direct binding of LIF to the membrane-bound form of gp130. In this study, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a M r of approximately 100,000, which is similar to that previously described (19Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar), and a previously undescribed small form with a M r of approximately 50,000. Both forms bound LIF specifically and were capable of forming heterotrimeric complexes with soluble hLIFRα in the presence of hLIF. In addition to the soluble forms of gp130, a soluble form of LIFRα was also detected in human urine and plasma. Escherichia coli-expressed hLIF (a gift from Sandoz Pharmaceutical Co., Hanover, Switzerland) was radioiodinated using a modified iodine monochloride method (24Contreras M.A. Bale W.F. Spar I.L. Methods Enzymol. 1983; 92: 277-292Crossref PubMed Scopus (76) Google Scholar). Anti-human gp130 monoclonal antibodies (mAbs), AM64, GPX22, and GPZ35, which were raised against Chinese hamster ovary cell-expressed extracellular domain of human gp130, were prepared as described previously (10Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1092) Google Scholar, 25Saito T. Taga T. Miki D. Futatsugi K. Yawata H. Kishimoto T. Yasukawa K. J. Immunol. Methods. 1993; 163: 217-223Crossref PubMed Scopus (44) Google Scholar). A goat anti-human LIFRα polyclonal antibody raised against the extracellular domain of human LIFRα was purchased from R & D Systems. A soluble form of human gp130 (shgp130), which consists of the Ig-like domain, hemopoietin domain, and three FN III modules, was expressed in the methylotropic yeast P. pastoris with a FLAGTM epitope tag (DYKDDDDK) at its N terminus and purified on an anti-FLAG M2 affinity column by elution with FLAG peptide as described previously (23Zhang J.G. Owczarek C.M. Ward L.D. Howlett G.J. Fabri L.J. Roberts B.A. Nicola N.A. Biochem. J. 1997; 325: 693-700Crossref PubMed Scopus (33) Google Scholar). A short form of soluble human gp130 (sshgp130) was made identically as shgp130 except that the construct lacked all three FN III modules. Protein quantitation for the purified samples was performed by amino acid analysis. To make a soluble form of hLIFRα, a cDNA encoding the hLIFRα (26Owczarek C.M. Layton M.J. Metcalf D. Lock P. Willson T.A. Gough N.M. Nicola N.A. EMBO J. 1993; 12: 3487-3495Crossref PubMed Scopus (41) Google Scholar) was altered at its 5′-end to encode an XhoI site and an in-frame 12CA5 epitope (YPYDVPDYA) (27Wilson I.A. Niman H.L. Houghten R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (655) Google Scholar). The sequence at the N terminus of the recombinant LIFRα was GAPYPYDVPDYA. The 3′-end was modified to encode an XbaI site and a stop codon was introduced after position 536 (2Gearing D. Thut C.J. VandenBos T. Gimpel S.D Delaney P.B. King J. Price V. Cosman D. Beckmann M.P. EMBO J. 1991; 10: 2839-2848Crossref PubMed Scopus (516) Google Scholar) so that the recombinant LIFRα only contained the two hemopoietin domains and the intervening Ig-like domain. The cDNA was subsequently cloned into the yeast expression vector pPIC9 and expressed in P. pastoris as described (23Zhang J.G. Owczarek C.M. Ward L.D. Howlett G.J. Fabri L.J. Roberts B.A. Nicola N.A. Biochem. J. 1997; 325: 693-700Crossref PubMed Scopus (33) Google Scholar). The protein was partially purified by gel filtration chromatography and quantified by Scatchard analysis of hLIF binding isotherms (17Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar). Male and female normal human urine was collected from volunteers after informed consent, and 0.02% (v/v) Tween 20 and 0.02% (w/v) sodium azide were added. Small scale urine concentration was carried out using a Centriprep-10 (Amicon), and large scale concentration was performed using the Sartorius EasyFlow Device with a cellulose triacetate membrane (molecular weight cut-off of 20,000). Any precipitating materials in urine occurring before or after concentration were removed by centrifugation. Concentrated human urine was applied to a Superdex 200 10/30 column (Amersham Pharmacia Biotech), previously equilibrated in 20 mm phosphate-buffered saline (0.15 m), pH 7.0, containing 0.02% (v/v) Tween 20 and 0.02% (w/v) sodium azide (PBS). Samples were eluted with PBS at a flow rate of 0.5 ml/min, and 0.5 ml fractions were collected. Aliquots of samples were incubated with125I-hLIF in the absence or presence of unlabeled hLIF or antibodies in a final volume of 15 μl for at least 1 h at 4 °C. Then 5 μl of a 12 mm solution of the bifunctional cross-linker BS3 (Pierce) in 20 mmphosphate-buffered saline (pH 7.0) was added, and the mixtures were incubated for 30 min at 4 °C. Samples were mixed with 7 μl of 4-fold concentrated SDS sample buffer and analyzed by either 7.5 or 10% SDS-PAGE under nonreducing conditions. The gels were dried and visualized by either autoradiography or PhosphorImager analysis (Molecular Dynamics). The rhLIF affinity column was prepared by covalently coupling 1 mg of E. coli-derived rhLIF to 1 ml of Affi-Gel 10 (Bio-Rad) according to the manufacturer's instructions. Normal human urine samples were concentrated 100-fold as described above and incubated with 1 ml of hLIF-Affi-Gel 10 resin for 3–5 h at 4 °C. After unbound proteins were removed by centrifugation, the hLIF affinity beads were washed with 16 × 1 ml of PBS followed by additional washes with 8 × 0.3 ml of 10-fold diluted Actisep elution medium (Sterogenes Bioseparations, CA). The bound protein was then eluted with 10 × 0.3 ml of undiluted Actisep elution medium. The affinity column eluates were buffer-exchanged into PBS using NAP-5 columns (Amersham Pharmacia Biotech). Using the same procedures, LIF-binding proteins were enriched and partially purified on the hLIF affinity column from 50 ml of an outdated normal human plasma sample (obtained from the Royal Melbourne Hospital Blood Bank). Aliquots of buffer-exchanged fractions were analyzed for their ability to bind to 125I-hLIF using the cross-linking protocol described above. Aliquots (20 μl) of the hLIF affinity column eluate, 0.2 μg/ml shLIFRα, or 2 μg/ml shgp130 were incubated with 125I-hLIF (800,000 cpm) in the absence or presence of 50 μg/ml unlabeled hLIF in a final volume of 50 μl for at least 1 h at 4 °C. Then 10 μl of a 12 mmBS3 solution was added, and the mixtures were incubated for 30 min at 4 °C. After adding 1 m Tris-HCl buffer (pH 7.5) to a final concentration of 50 mm, the cross-linking reactions were incubated for 40 min at room temperature. The cross-linked samples were then mixed with an anti-human LIFRα polyclonal antibody at a concentration of 50 μg/ml. After a 30-min incubation at 4 °C, the mixtures were added to 30 μl of 50% (v/v) protein G-Sepharose gel slurry (Amersham Pharmacia Biotech) previously equilibrated in PBS and incubated for 30 min at 4 °C. The samples were centrifuged, and the protein G-Sepharose beads were washed with 4 × 0.5 ml of PBS. For elution, the beads were mixed with 30 μl of 2× concentrated SDS sample buffer. The supernatants were then analyzed by 7.5% SDS-PAGE under nonreducing conditions. Protein concentrations of pure recombinant soluble receptors were determined by amino acid analysis on a Beckman 6300 high performance amino acid analyzer equipped with a model 7000 data analyzer (Beckman). M1 cells (∼107 cells/sample) were stimulated for 5 min at 37 °C with either 1 ng of hLIF or saline together with either soluble hLIF receptor or soluble hgp130 and then lysed in 50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 2 mm EDTA, 1% Triton X-100, 2 mm NaF, 1 mm Na3VO4, and proteinase inhibitors. After pelleting insoluble material and protein standardization, approximately 100 μg of total cellular proteins were subjected to 4–15% acrylamide SDS-PAGE under reducing conditions and then transferred to a prewetted polyvinylidene difluoride membrane (PVDF-Plus, Micron Separations Inc.). After blocking, the membrane was incubated with an anti-phospho-STAT-3 polyclonal antibody (New England Biolabs), followed by incubation with a goat anti-rabbit Ig polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark). The phosphorylated STAT-3 protein was visualized by radiography using the ECL system (Amersham Pharmacia Biotech). To check the quantity of protein loading, the same membranes were stripped with 0.1m glycine-HCl, pH 3.0, for 30–60 min and washed three times in PBS, 0.1% Tween 20 before reprobing with a rabbit polyclonal antibody to STAT-3 (K-15, Santa Cruz Biotechnology, Inc.). To quantify the soluble LIFRα, an aliquot of an outdated normal human plasma sample (obtained from the Royal Melbourne Hospital Blood Bank) was first precleared with protein G-Sepharose beads at a ratio of 1:0.2 (v/v) for 1 h at 4 °C. The protein G-absorbed plasma was then incubated in the presence or absence of 2 μg of a goat anti-human LIFRα polyclonal antibody for 1 h at 4 °C, followed by the addition of 25 μl of protein G-Sepharose beads and a 2-h incubation at 4 °C. Immunoprecipitation of recombinant shLIFRα at various concentrations was performed in parallel except that the preabsorption step with protein G beads was not included. The immunocomplexes were washed with 3 × 1 ml of PBS containing 0.02% (v/v) Tween 20 and eluted from the protein G beads by boiling in SDS sample buffer under reducing conditions for 5 min before being subjected to 7.5% acrylamide SDS-PAGE. The Western blotting was performed as described above except that the anti-human LIFRα polyclonal antibody and a rabbit anti-goat Ig polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark) were used as the first and second antibodies, respectively. Human urine samples, collected from six healthy individuals (H1–H6) were concentrated and tested for soluble LIF-binding proteins by chemical cross-linking. Analysis of the cross-linking products by SDS-PAGE (Fig. 1) indicated that 125I-hLIF was cross-linked specifically to two species of proteins in all six samples with M r of approximately 100,000 (here referred to as the “large form”) and 50,000 (here referred to as the “small form”) after subtraction for the M r of the bound unglycosylated hLIF, respectively. The levels of the two hLIF-binding proteins varied in the six samples. This variation was likely to be due to the differences in protein content of these samples (data not shown). To examine whether the two hLIF-binding proteins were part of a preformed complex in urine, concentrated human urine was fractionated on a Superdex 200 gel filtration column as shown in Fig. 2 A, and fractions were then analyzed for 125I-hLIF binding by chemical cross-linking. Analysis of column fractions 21, 23, 25, 27, 29, and 31 by SDS-PAGE (Fig. 2 B) after cross-linking showed that the two hLIF-binding proteins were completely separated from each other according to their sizes, suggesting that they do not exist in a preformed complex in human urine. The M restimates of the two proteins by gel filtration were consistent with those obtained above. Also, it can be seen from Fig. 2 B that there was a downward trend in M r for both the large and small forms of the LIF-binding proteins across the fractions being assayed. This may be due to differential glycosylation of the two proteins. In mouse and human serum, the presence of soluble forms of the LIF receptor components, mouse LIFRα and human gp130, respectively, has been described (17Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar, 18Yamaguchi-Yamamoto Y. Tomida M. Hozumi M. Leukemia Res. 1993; 17: 515-522Crossref PubMed Scopus (15) Google Scholar, 19Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). It has also been demonstrated that LIF can bind to gp130 directly (22Modrell B. Liu J. Miller H. Shoyab M. Growth Factors. 1994; 11: 81-91Crossref PubMed Scopus (19) Google Scholar, 23Zhang J.G. Owczarek C.M. Ward L.D. Howlett G.J. Fabri L.J. Roberts B.A. Nicola N.A. Biochem. J. 1997; 325: 693-700Crossref PubMed Scopus (33) Google Scholar), although the affinity was relatively low (23Zhang J.G. Owczarek C.M. Ward L.D. Howlett G.J. Fabri L.J. Roberts B.A. Nicola N.A. Biochem. J. 1997; 325: 693-700Crossref PubMed Scopus (33) Google Scholar, 28Hudson K.R. Vernallis A.B. Heath J.K. J. Biol. Chem. 1996; 271: 11971-11978Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To examine whether the detected125I-hLIF binding activity in human urine was due to the presence of these reported proteins, we first performed competitive cross-linking experiments, as shown in Fig. 3, in which 125I-hLIF was mixed with increasing concentrations of unlabeled hLIF prior to cross-linking to a partially purified urinary LIF-binding protein sample (Fig. 3 A). This was then compared with the same cross-linking to (a) a recombinant form of soluble human gp130 (shgp130; Fig. 3 B), which consists of the Ig-like domain, hemopoietin domain, and three FN III modules with a FLAGTM epitope tag at the N terminus, (b) a recombinant short form of soluble human gp130 (sshgp130; Fig. 3 C) identical to shgp130 except that the construct lacked the FN III modules, or (c) a recombinant form of soluble human LIFRα (shLIFRα; Fig. 3 D). Densitometric analyses of these data (not shown) revealed that half-maximal inhibition of125I-hLIF cross-linking to both the large and small forms of the two urinary binding proteins occurred at approximately 750 ng/ml unlabeled hLIF, similar to the hLIF concentrations required to inhibit 50% of the cross-linking to both shgp130 and sshgp130, whereas an approximately 3-fold smaller amount of hLIF was required to achieve the same inhibition for shLIFRα. These results indicated that the relative binding affinities of 125I-hLIF to the two forms of urinary binding proteins were similar to those of125I-hLIF to recombinant shgp130 and sshgp130, suggesting that the LIF binding activity in human urine might be due to the presence of soluble forms of gp130 with different truncations at the C-terminal ends. To formally test this possibility, the urinary LIF-binding proteins were first purified by a hLIF affinity chromatography step as described under “Experimental Procedures.” The eluates from the hLIF affinity column were then fractionated on a Superdex 200 gel filtration column to separate the large and small forms of the LIF-binding proteins (data not shown). The appropriate fractions were subsequently analyzed in cross-linking experiments, in which 125I-hLIF was incubated with the purified large or small form of the urinary LIF-binding protein in the presence of anti-shgp130 mAbs prior to cross-linking. As shown in Fig. 4, cross-linking in the presence of mAb AM64 yielded a higher M r complex in addition to the 125I-hLIF·large form complex (Fig. 4 A, compare lanes 6 and 8) without a significant effect on 125I-hLIF cross-linking to the small form (Fig. 4 B, compare lanes 6 and 8), whereas the addition of mAb GPZ35 in the cross-linking mixture generated higher M r complexes in both cases (Fig. 4 A and B, lanes 10). In contrast, mAb GPX22 completely inhibited the cross-linking of125I-hLIF to the small form (Fig. 4 B, compare lanes 6 and 9) but only partially inhibited the125I-hLIF cross-linking to the large form (Fig. 4 A, compare lanes 6 and 9). For comparison, these mAbs were also added to the cross-linking mixtures of recombinant shgp130, sshgp130, and shLIFRα. As before, mAb AM64 only affected shgp130 (Fig. 4 A, lane 3) but not sshgp130 (Fig. 4 B, lane 3) cross-linking to125I-hLIF. Unexpectedly, mAb GPZ35 did not significantly affect the cross-linking of 125I-hLIF to either shgp130 (Fig. 4 A, lane 5) or sshgp130 (Fig. 4 B, lane 5) despite being able to produce a higher M r species when added to the cross-linking mixture of the small form (Fig. 4 B, lane 10). This may be due to the possibility that mAb GPZ35 could not recognize Pichia-expressed gp130, suggesting that it recognizes a carbohydrate-related epitope. Alternatively, it may suggest that the FLAG peptide tagged at the N termini of the two recombinant Pichia-gp130 proteins somehow affects the GPZ35 binding. In the case of mAb GPX22, it almost completely inhibited the cross-linking of 125I-hLIF to both shgp130 (Fig. 4 A, lane 4) and sshgp130 (Fig. 4 B,lane 4). As expected, these mAbs showed little effect on the cross-linking to shLIFRα (Fig. 4 A, lanes 13–15). By themselves, these mABs did not significantly cross-link to 125I-hLIF (Fig. 4 B, lanes 11–13). Taken together, these results revealed that the small form of the urinary LIF-binding protein was a truncated form of gp130 probably containing only the hemopoietin domain but lacking all or almost all three FN III modules and that the large urinary LIF-binding form contained gp130. Meanwhile, two observations from the antibody analysis experiments also suggested that the LIF binding activity in the purified large form fraction was not entirely due to the presence of soluble gp130. First, mAB AM64 was able to almost completely shift the125I-hLIF·shgp130 complex to a higher M r species (Fig. 4 A, lane 3) but only partially did so to the large form (Fig. 4 A, lane 8). Second, mAb GPX22 could completely inhibit the cross-linking of 125I-hLIF to the recombinant shgp130 (Fig. 4 A, lane 4) but could only partially inhibit the 125I-hLIF cross-linking to the large form (Fig. 4 A, lane 9). One possibility was that the purified large form fraction also contained the soluble LIFRα component. Evidence for this also came from a large scale purification (10 liters of human urine were used) of the urinary LIF-binding proteins using hLIF affinity chromatography described under “Experimental Procedures” (data not shown). When eluates from the affinity column were analyzed in cross-linking experiments, the fraction containing the most LIF-binding activity showed an extra higher M r species in addition to the normal large and small forms (Fig. 5, lane 8). To test this forma" @default.
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• W2093643472 title "Identification and Characterization of Two Distinct Truncated Forms of gp130 and a Soluble Form of Leukemia Inhibitory Factor Receptor α-Chain in Normal Human Urine and Plasma" @default.
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